Disinfestation and disinfection of food, perishables and other commodities

ABSTRACT

A method and system for disinfecting and disinfesting a commodity, such as a perishable agricultural commodity, by treatment with an environment of low oxygen/high ballast gas with cycled pressure changes that overwhelm and damage the respiratory system of the insect without damaging the host commodity. The system and method may also include the introduction of disinfectants, antiseptics and other toxic chemicals or the exposure to radio frequencies with intense electric fields that may increase the metabolic activity of the pest or decrease the fitness of the pest within the low oxygen environment. Treatments according to the methods can also increase the shelf life of agricultural commodities by eradicating or delaying the growth of bacteria, fungi, protozoa and other microbial pests.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/429,647 filed on May 5, 2006, now U.S. Pat. No. 7,975,427,incorporated herein by reference in its entirety, which is acontinuation of PCT international application serial numberPCT/US2004/013225, filed on Apr. 30, 2004, incorporated herein byreference in its entirety, which claims priority from U.S. provisionalpatent application Ser. No. 60/517,806 filed on Nov. 5, 2003,incorporated herein by reference in its entirety. Priority is claimed toeach of the foregoing applications.

The above-referenced PCT international application was published on May26, 2005 as PCT International Publication No. WO 2005/046743,incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable

NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION

A portion of the material in this patent document is subject tocopyright protection under the copyright laws of the United States andof other countries. The owner of the copyright rights has no objectionto the facsimile reproduction by anyone of the patent document or thepatent disclosure, as it appears in the United States Patent andTrademark Office publicly available file or records, but otherwisereserves all copyright rights whatsoever. The copyright owner does nothereby waive any of its rights to have this patent document maintainedin secrecy, including without limitation its rights pursuant to 37C.F.R. §1.14.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains generally to insect and other pest controltreatments and more particularly to methods for rapidly eliminatinginfestations of insects and other living organisms from commoditiesusing low oxygen atmospheres, pressure differentials and metabolismmanipulations with disinfectants, antiseptics, toxics or exposure tointense non-thermal radio frequencies.

2. Description of the Related Art

Every year considerable quantities of pesticides are applied tocommodities by producers at various stages of agricultural production,from pre-planting to post harvest, in order to eradicate unwantedinsects and other animal, microbial and fungal pests. The presence ofegg, larval and adult forms of insects creates the possibility ofcross-infestation of commodities and increasing losses duringtransportation and storage. Established quarantine barriers regulatetransportation of agricultural commodities worldwide in order to reducethe potential for propagation and transportation of non-indigenouspests. Many commodities cannot be legally imported or exported tovarious countries without pesticide treatments to eliminate quarantinepests and to certify that the commodities are free from pests.

Methyl bromide, for example is widely used in the industry as a gaseousfumigant that can disinfest a variety of fresh foods, agricultural soilsand structural facilities. However, methyl bromide is scheduled to bebanned in the next few years because of the capability of methyl bromideto scavenge ozone in the atmosphere. Agriculture in the United Statesused about 60 million pounds of methyl bromide before the mandatoryreductions began in 1999.

The use of methyl bromide or other chemicals in the fresh fruit industryis often unsatisfactory due to the creation of cosmetic blemishes or areduction in the effective shelf life of the fruit. In addition,applications of methyl bromide at concentrations sufficient to controlpests on stored and exported commodities may produce bromide residuelevels that are relatively high.

Likewise, other pesticides known in the art have shown erraticperformance at low concentrations and have produced crop damage andunacceptable residue levels in some cases. Other pesticides that arewidely used with pre-harvest and post-harvest applications includephosphine, chloropicrin, 1,3-dichloropropene, Telone/Vapam, sulfarylfluoride and hydrogen cyanide. The use of pesticides in general andthese insecticides in particular are of global concern due to thedetrimental effects they have on animals, air, water and soil as well asthe impact they have on public health and agricultural workers.

Another approach to the eradication of insect infestation in foodcommodities in the art is the use of thermal energy. However, thermalenergy, such as the use of hot water, is unsatisfactory because it cancause rapid deterioration of the commodity and typically uses a largeamount of energy. Thermal energy is normally used in the fresh produceindustry only when no other alternatives are viable or available.

A further approach to disinfestation has been to expose the commodity toa controlled atmosphere with low oxygen concentrations and increasedcarbon dioxide concentrations. However, these techniques have beeninconsistent and often ineffective for eliminating insects, mites andother pests. Present controlled atmosphere approaches require severaldays to weeks to conduct and are therefore of limited use in the freshproduce industry. One reason that controlled atmosphere techniques areonly marginally effective is that many insects can survive low oxygen orincreased carbon dioxide environments by collapsing portions of theirrespiratory system to form air sacks providing a reserve of oxygen. Suchreserves may allow some types of insects to survive for several days ormore.

Furthermore, some fresh foods may experience detrimental changes incolor, texture, acidity and other characteristics from prolongedexposure to high carbon dioxide environments. Accordingly, controlledatmosphere techniques are not considered viable for disinfestation andquarantine applications with fresh produce.

Additionally, there are major human safety concerns that exist todayfrom the potential contamination of food commodities with pathogenicbacteria such as Escherichia coli O157:H7, Salmonella sp., Listeria, andespecially Campylobacter. Each of these pathogenic bacteria has recentlybeen identified as disease causing agents from the consumption of manycommon food commodities. It is estimated that outbreaks of food borneillnesses in the United States affect 12 million people and result inthe death of approximately 4,000 individuals annually. Similarly, thecontrol of protozoa (i.e. Toxoplasma sp., Cryptosporidium sp., and/orCyclospora sp.) and other parasites on many foods and especially onfresh fruits and vegetables is an important challenge for agriculturalproducers. For example, humans may become infected with parasites byingesting tissue cysts from undercooked meat or other infected food orwater. Recently, several outbreaks of Cyclospora associatedgastroenteritis in humans were linked to the consumption of raspberries,lettuce and basil. There are presently no practical methods available todisinfect foods from infective oocysts.

Microbial activity may also generate a variety of toxins, such asAflatoxin from Aspergillius flavus in grains, that are detrimental topublic health or may otherwise make the commodity lose its value in themarketplace. Agricultural commodities such as fresh produce, grains,seeds, and spices may also be affected by fungal and/or bacterialcontaminants. It is therefore desirable to inhibit the presence ofdisease-carrying organisms within food and agricultural commodities aswell as eradicate insect infestations. This can be accomplished byeither slowing down the development of spoilage organisms (biostaticeffects) or by casing a lethal effect on the organism (biocidal effect).

Accordingly, there is a need for an apparatus and method for eradicatinga wide variety of insect and microbial pests that is effective and doesnot leave toxic residues or alter the characteristics of the commoditythat is treated. The present invention satisfies these needs as well asothers and generally overcomes the deficiencies in the art.

BRIEF SUMMARY OF THE INVENTION

The present invention generally provides a method for rapid, quarantinelevel disinfestation of insects, mites and other biological pests from avariety of commodities such as fresh, dried or processed foods as wellas cut flowers, soils and historical artifacts and the like. The methodmay also provide biostatic effects (i.e. delays of microbial growth) orbiocidal effects on microbes such as protozoa, bacteria and fungi thatare often responsible for reducing the shelf life or storage life of acommodity.

The method is based on sequestering the food commodity for a period oftime, typically less than 24 hours and often 1 to 6 hours, in acontainer preferably containing an environment with extremely low oxygen(preferably less than 0.01%) and a high carbon dioxide (preferablygreater than 99.9%) levels, at above or below barometric pressure. Themethod includes an initial pressure cycling procedure that is designedto manipulate the respiratory system of the insects in order to overcometheir ability to establish a reserve of air within collapsible air sacs.The mechanical forces applied to the respiratory system of the pests bythe cyclic pressure differential may also damage the tubular respiratorynetwork. Such damage reduces the overall capacity of the respiratorysystem to exchange gases as well as to increase the sensitivity of theinsect pest to low oxygen or high carbon dioxide conditions. Theduration of treatment, the number of cyclic repetitions and the size ofthe pressure differential can be adapted to the sensitivity of theinsect pest and the commodity to anoxic environments. In this manner,mortality effects are maximized and pest survival is greatly reducedover time allowing the procedure to be completed in the range of lessthan 1 hour to 24 hours.

The gaseous environment used for disinfestation may also inducesimultaneous microbial eradication or growth delay effects for 2-3 daysand longer at room temperature. The growth delay effects can be extendedeffectively to 8-10 days or more under refrigerated storage. Due to theshort time required for effective disinfestation and the non-toxicnature of the gases involved, the potential for sensory and/orphysiological changes in the host commodity (i.e. due to anaerobicrespiration or fermentation) are largely avoided. In addition, theshortened time makes the method a practical and economical approach overprevious attempts to use modified atmospheres for disinfestation anddisinfection of foods and fresh horticultural perishable commodities.

In one embodiment, a method is provided that includes sequestering ahost commodity to be treated in a container or enclosure for a period oftime less than approximately 24 hours. An environment is created in thecontainer that preferably contains less than approximately 0.01%) oxygenand approximately 99.9% ballast gas or gases (i.e. carbon dioxide,nitrogen, others). Although very low oxygen conditions are preferred,higher levels of oxygen can be used. The environment is maintained atabove or below barometric pressure. In one embodiment, the enclosure isconfigured to permit the interior to be maintained at below the ambientbarometric pressure. In another, the environment is maintained at levelsabove the ambient barometric pressure. A further embodiment provides acontainer that can expose a commodity to atmospheres that cycle frombelow to above or from above to below ambient barometric pressures etc.for one or more cycles.

The initial cycling of positive and negative pressures designed tomanipulate the insect's respiratory system may also be repeatedperiodically in order to vent any detrimental gases formed frommetabolic processes in fruits and vegetables so as to avoid or minimizethe potential for physiological changes including anaerobic fermentationwhen overall treatment times extend to several hours.

The use of carbon dioxide or nitrogen as a ballast gas at normal orslightly greater barometric pressures may also induce changes in thecellular metabolism of the insects, mites or microbial contaminants. Forexample, the use of carbon dioxide can change the acid-base equilibriumor displace the oxygen equilibrium within the respiratory system andbody of the insect pest.

In order to accelerate mortality and other biological effects on insectsand microbes, additional metabolic stress may be applied to the pests.In one embodiment, further metabolic stress may be generated through theuse of an oscillating radio frequency field with appropriate electricfield intensities. The interaction of RF generated electric fields withinsects and mites cause increased oxygen demands because the conductiveinsects attempt to physically align themselves to rapidly changing fieldorientations. The radio frequency fields may also have a direct effecton the fitness of the insects and other pests by weakening and damagingthe insects and reducing their tolerance of the anoxic environment.

In another embodiment, metabolically toxic disinfectants like ozone orhydrogen peroxide and/or antiseptics such as ethanol may be quicklyevaporated within the container or added externally into a reducedpressure environment to allow for their rapid gasification and acomplete distribution over the commodity's surface. The use of suchdisinfectants not only assists with disinfestation, it has the addedbenefit of providing disinfection of the commodity as well.

Alternatively, in another embodiment, oxygen radicals and ozone can beproduced in situ within the commodity container using oscillating orpulsed radio frequency fields. Ozone and oxygen radicals may also beintroduced from an external source. Oxygen radicals and ozone in smallquantities induce toxic effects to cellular metabolism as well asmetabolic stress to an insect pest.

In another embodiment, the environment in the container includes toxicgases or regulated pesticides in addition to the low oxygen and highballast gas environment. Gases or vapors that are toxic to insect pests,such as nitrogen oxide and sulfur oxide gases and propylene oxide, mayalso be used to decrease the fitness and increase the metabolic stresson an insect pest during treatment. In some applications, the method mayuse insecticides, fungicides or bactericides that have a known effect ona particular pest. When used with the low oxygen environment alone or incombination with other stress producing agents, the quantity ofpesticide that is required to be used will be less than the quantitythat is required to eradicate the pest directly. Accordingly, residuesof such pesticides left on the commodity will be negligible or wellwithin acceptable limits.

It can be seen that disinfestation and disinfection effects may becombined and maximized. The combination of the effects of an environmentwith a low oxygen, high ballast gas atmosphere along with the cycledpressure differential and metabolic stressors allows for virtuallycomplete disinfestation of the commodity. The modified gaseousenvironment used may also simultaneously induce biocidal effects ormicrobial growth delay effects lasting a few days or more at roomtemperature thereby increasing the shelf life of commodity. Thesemicrobial control effects can be extended effectively many times longerwith the use of refrigerated storage for the commodities.

The shortened time required for effective disinfestation anddisinfection, and the use of natural, transient chemicals in theprocedure, eliminates or minimizes the potential for sensory and/orphysiological changes in the host commodity (i.e. due to anaerobicrespiration or fermentation), while the production of residues islargely avoided. In addition, the shortened time makes this method apractical and economical approach over previous attempts to use standardmodified atmospheres in foods and in fresh horticultural perishablecommodities. Design, engineering, and manufacturing of large, commercialsize systems constructed with special materials and functionalcapabilities are now possible.

According to one aspect of the invention, a method is provided fordisinfestation and disinfection of a commodity by depriving an insect ormite of oxygen and introducing metabolic stress in said insect or mitewhile in an oxygen deprived state.

According to another aspect of the invention a method for controlling aninsect, mite or other biological pest is provided comprising exposing apest to a reduced oxygen environment for a period of time; manipulatingthe respiratory system of the pest in order to overcome the ability ofthe pest to establish a reserve of air within collapsible air sacs andthen exposing the pest to at least one chemical shown to cause metabolicstress in insects, mites and other biological pests.

Another aspect of the invention provides a method for microbial control,comprising the steps of exposing microbes to an environment of lowoxygen high ballast gas concentrations for a period of time and exposingthe microbes to at least one metabolically toxic agent while exposed tothe anoxic environment.

According to another aspect of the invention, a method is provided fordepriving an insect or mite of oxygen and then introducing metabolicstress in the insect or mite while in an oxygen deprived state bysubjecting the insects or mites to an oscillating radiofrequency field.

According to another aspect of the invention, a method for controllingan insect, mite or other biological pest is provided that includesexposing a pest to a reduced oxygen environment for a period of time;manipulating the respiratory system of said pest; exposing said pest toa radio frequency field for a period of time and exposing the pest to atleast one chemical shown to cause metabolic stress in insects, mites andother biological pests.

An object of the invention is to provide an effective and economicalmethod for disinfestation and disinfection of a commodity that isnon-thermal, residue free and a legitimate alternative to methyl bromidefumigation.

A further object of the invention is to provide an effective andinexpensive method for controlling protozoa, parasites and tissue cystinfectious agents.

Another object of the invention is to provide a method of disinfestationthat overcomes the ability of insects, mites and other pests to createand maintain a reserve of air in the respiratory system.

Another object of the invention is to provide a method to applymechanical forces on the respiratory system of a pest that forces therelease of stored air and damages the components of the respiratorysystem while leaving the host commodity undamaged.

Still another object of the invention is to provide a method ofsimultaneously disinfesting and disinfecting heat sensitive commoditiescontinuously or in batches.

Another object of the invention is to provide a method of disinfestationand disinfection that displaces the acid-base balance in cells withcarbon dioxide to lower the pH and cause lethal or sub-lethal effects inthe cells and tissues of the pest.

A further object of the invention is to provide a pressure cycled anoxicenvironment that may also include secondary stress effects with the useof toxic gases, volatile disinfectants or antiseptics to increase theeffectiveness of anoxic environment on the pest.

Another object of the invention is to provide secondary stress effectswith the use of radio frequencies in oscillating electric fields to bothincrease the oxygen demands in insects and mites and to generate atomicoxygen and molecular ozone in situ within the enclosed environment.

Another object of the invention is to provide a method of disinfestationand disinfection that has a short duration and can be adapted to treatperishable commodities without changing the chemical properties, thephysical characteristics or shortening the shelf life of the commodity.

Another object of the invention is to provide an apparatus and methodfor disinfestation and disinfection of commodities that is simple touse, easy to construct and inexpensive to purchase and maintain.

Further objects and advantages of the invention will be brought out inthe following portions of the specification, wherein the detaileddescription is for the purpose of fully disclosing preferred embodimentsof the invention without placing limitations thereon.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The invention will be more fully understood by reference to thefollowing drawings which are for illustrative purposes only:

FIG. 1 is a flow diagram of one embodiment of the disinfestation methodaccording to the present invention.

FIG. 2 is a flow diagram of one embodiment of the disinfection methodaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Referring more specifically to the drawings, for illustrative purposesthe present invention is embodied in the apparatus and method generallyshown in FIG. 1 through FIG. 2. It will be appreciated that theapparatus mentioned may vary as to configuration and as to details ofthe parts, and that the method may vary as to the specific steps andsequence, without departing from the basic concepts as disclosed herein.

A wide variety of commodities may be subject to infestations of insectsand other pests during packing, shipment and storage. The methods of thepresent invention may be particularly beneficial to the disinfestationand disinfection of fresh produce including fruits and vegetables aswell as dried foods including nuts; grains; seeds; dehydrated foodsincluding cereals; processed foods; animal feeds; eggs; ornamental andcut flowers; wood products; nursery stocks; agricultural soils includingnursery or containerized soils and non food commodities needingdisinfestation such as archeological and art objects. Perishable foodcommodities are often the most difficult to disinfest in the art and areused for illustration of the methods of the present invention.

Agricultural commodities are typically boxed in the field or taken fromthe fields and packed in a packing plant or stored in a storage facilitybefore being transported to market. Insects and other pests may bepresent on a commodity in the form of adults as well as egg or larvalforms. Quarantine level disinfestation requires eradication of everyform of the insect to guarantee that no further pests or damage willappear during transportation and storage of the commodity.

The apparatus and methods of the invention are an effective alternativeto methyl bromide that does not leave any toxic residues or damage thecosmetic appearance of the commodity. The methods can also lengthen theshelf life of many commodities in addition to eradicating the biologicalpests.

Turning now to FIG. 1, a flow diagram of one embodiment 100 of themethod for disinfestation of a commodity is generally shown. At block110, the commodity is sequestered in an enclosure or container. Thecontainer or enclosure can be sized and adapted to the type of commoditythat is to be treated from single flats to multiple pallets ofcontainers. The container or enclosure is preferably gas tight andconfigured to remain sealed during positive or negative pressure changeswithin the interior of the container. In one embodiment, the containerhas intake and output valves to permit the efficient evacuation anddeposition of gasses within the container. In another embodiment, theenclosure is configured to receive multiple pallets and may be roomsized. In this embodiment, there may be multiple valves connected to theenclosure to efficiently evacuate and transfer gas or air from theinterior of the enclosure as well as manipulate the interior pressure ofthe enclosure.

At block 120 of FIG. 1, the sequestered commodity is exposed to ananoxic environment for a period of time. The environment that isestablished in the container preferably has a very low oxygen content toeliminate the source of diffusible oxygen for cellular respiration ininsects, mites and microbes etc. It is also preferred that the createdenvironment have a high ballast gas content. In addition, a cycledpressure differential is created within the container for one or morecycles over a period of time to apply mechanical stresses on therespiratory system of the infesting insects.

The type of pest that is to be eradicated and the sensitivity of thecommodity to the ballast gases and pressure changes govern the time ofexposure to the anoxic environment. The time of exposure is typicallyless than 24 hours and usually in the range of 1 to 12 hours. Thecomparatively short treatment times will not pose a significant delay inbringing fresh fruits or vegetables to market. It will be also seen thattreated commodities will typically have a greater market life thanuntreated commodities.

It is preferred that an extremely low oxygen environment of less than 1%oxygen per volume be created in the container or enclosure. Anenvironment of less than approximately 0.01% oxygen per volume isparticularly preferred. Oxygen is an essential metabolite for allinsects and mites as well as for aerobic microbes and it must beavailable in the cellular environment in order to sustain vitalmetabolic activity. All insects and mites, and many microbes, areaerobic organisms utilizing glycolysis, the Kreb's cycle, andelectron-transport mechanisms for metabolic reactions leading to theconversion of nutrients (i.e. sugars) into stored chemical energy (ATP).Mortal injuries may be selectively inflicted on pests by preventing theuptake of oxygen and restricting the discharge of carbon dioxide throughthe respiratory system of the insects while leaving the host commodityunaffected.

The preferred ballast gases are carbon dioxide, nitrogen gas or acombination of carbon dioxide and nitrogen gases. While carbon dioxideor nitrogen gases are preferred, other gases other than oxygen that donot react significantly with the host commodity can be used. It ispreferred that the ballast gas in the environment of the container havegreater than approximately 99% carbon dioxide or nitrogen gas by volume.A ballast gas concentration of greater than approximately 99.9% byvolume is particularly preferred. The use of a high concentration of aballast gas or combination of gases like carbon dioxide or nitrogendisrupts the flow of any remaining oxygen in the respiratory tract ofthe insect and may influence the outward exchange of carbon dioxide fromthe respiratory tissues.

A pressure differential is preferably established with the pressurechanges in the environment of the enclosure conducted over one or morecycles. The pressure differential cycles may have a range that iscompletely hyperbaric, completely hypobaric or a range that ishyperbaric on one extreme and hypobaric on the other. In one embodiment,the pressure differential ranges from −10 to +5 pounds per square inchfrom the ambient barometric pressure baseline. In another embodiment,the environment is maintained at pressures above the ambient barometricpressure with the pressure differential ranging from +1 to +8 psi. Inanother embodiment, the container environment is maintained at pressuresbelow the ambient barometric pressure with the pressure differentialranging from −7 to −2 psi. Although these pressure ranges may bepreferred, it will be understood that any pressure range can be usedthat does not damage the commodity and demonstrates a physiologicaleffect on insects, mites and other animals.

The pressure cycles may be conducted after the low oxygen, high ballastgas environment is established in the container. Alternatively, thepressure cycles may begin with the initial purging of the air in thecontainer as the low oxygen environment is established in the container.In this embodiment, the air within the container is preferably removedwith several consecutive or time delayed cycles and replaced withballast gasses to provide an environment that is preferably less thanone percent oxygen by volume.

It can be seen that the cycling pressure differential in an establishedenvironment or the progression of low pressure (vacuum) and highpressure purging of the environment within the container will eliminatethe ability of the insects to establish an air reserve with collapsiblesacs in the respiratory system. The airflow in the typical insectrespiratory system is controlled in part by muscles that operate flaplike valves within each tracheal trunk and spiracle, the opening in theexoskeleton of the insect. In low oxygen or other stress environments,sections of the system of tracheal tubes can collapse to form pocketsand provide the insect with a reserve of air. Positive and negativechanges in pressure can overcome the air reserve system of the insect sothat the insect will not be able to withstand exposure to the lowoxygen/high carbon dioxide environment. Mechanical forces from thelow-pressure conditions force open the collapsed air sacks in therespiratory system of the insect because of the difference in pressurein the respiratory system with respect to the pressure in the containerenvironment. Forced release of any stored air in the respiratory systemwill allow the air reserve of the insect to be replaced with a ballastgas thereby upsetting the oxygen equilibrium within the body of theinsect.

In addition, during the opposite cycle with increased pressure, gaseousoxygen in the remaining air is diluted with pressurized CO₂ and outsideforces are created on the spiral cuticles (taenidia) that providephysical support to the connecting tubes of the respiratory systemallowing them to remain open or avoid collapse.

Multiple hypobaric/hyperbaric (i.e. vacuum/pressure) cycles arepreferably utilized to remove the ambient oxygen in the container and toremove the air reserve in insects and mites as well as provide otherphysical and chemical stresses on insects while avoiding changes inquality attributes in the host commodity. For example, lower pressuredifferentials that do not to exceed approximately ±5 psi for shortdurations can be utilized to minimize potential physical (i.e. texture)damage to sensitive commodities such as wild berries and the like. Othercommodities that are not pressure sensitive may have larger pressuredifferentials than 5 psi with fewer cycles to achieve the desiredresults.

The amount of oxygen remaining in the entire insect respiratory system(both gaseous and dissolved) is diluted considerably with each pressurecycle according to the principles of Dalton's Law of Partial Pressures.The addition of carbon dioxide or nitrogen ballast gas to the containermay also induce changes in the cellular metabolism of the insects ormites. For example the use of carbon dioxide environments may result inchanges in the acid-base equilibrium (i.e. pH changes) in cells as wellas modify the dissolved/gaseous oxygen balance in the respiratorysystem. The displacement of the equilibrium between the dissolved oxygenin the insect body and the gaseous oxygen in the respiratory systemallows for the nearly complete removal of respiratory oxygen from theinsect causing toxic conditions in the body. Similar structural andbiological effects are observed in egg, pupae and larval forms ofinsects or mites.

The commodity is preferably kept in the anoxic environment for a periodof time, usually less than 24 hours. Between pressure cycles thecontainer is preferably maintained at or near the barometric pressure ofthe surroundings to eliminate the potential for the flow of air into thecontainer or gas from the container. A purging cycle of the environmentmay also be repeated periodically in order to vent any detrimental gasesthat are formed from metabolic processes in fruits and vegetables inorder to minimize the potential for physiological changes in thecommodity including anaerobic fermentation. The need for such venting ofgases will vary with the characteristics of the commodity.

In order to accelerate mortality and other biological effects oninsects, mites and other biological pests, additional secondary effectsmay be optionally initiated at block 130 of FIG. 1. Stimulated metabolicactivity and oxygen demand as well as secondary stresses on the insectsand mites accentuate the effect of the anoxic environment and increasesmortality. Multiple stressors may produce essentially complete insectmortality as well as reduce the time of exposure of the commodity to thecontrolled environment. The source of the secondary stress need not betoxic to the insects or mites or delivered at toxic concentrations.However, the combination of an anoxic environment and toxic materialsmay allow some toxins to be effective at very small concentrations. Itis preferred that the sources of the secondary effects leave little orno residues on the commodity.

Secondary effects using metabolically toxic disinfectants such as ozoneor hydrogen peroxide that are known to be effective with a specific pestmay be created at block 140. Hydrogen peroxide, for example, is anoptional disinfectant that can provide metabolic stress to insects andmites in relatively low concentrations leading to mortality whilesimultaneously inducing disinfection effects. The addition and dispersalof H₂O₂ in the enclosure takes advantage of the volatility of hydrogenperoxide in low-pressure conditions (p_(p)=1 mm Hg at 15° C.).

Likewise, antiseptics that can be quickly evaporated within thecontainer or added externally into a reduced pressure environment toallow for their rapid gasification and a complete distribution over thecommodity's surface may be added at block 150 of FIG. 1. Althoughvolatile antiseptics and a low pressure environment are preferred, itwill be understood that any antiseptic and a neutral or positivepressure environment can be used.

One chemical that has been shown to be effectively dispersed in alow-pressure environment is ethanol. Ethanol (EtOH) and other alcoholsare known antiseptics and their action is rapid and effective,especially for plant pathogens like fungi and for human pathogens suchas bacteria. Ethanol vapors can also be generated rapidly within acontainer or be added into a low-pressure container by spraying or maybe introduced in the form of vapor by heating to permit rapid anduniform dispersal. Partial pressures of approximately 40 mm of Hg arepossible with ethanol in a reduced pressure environment at 15-20° C.Spraying ethanol through nozzles or simply adiabatically expanding hot,liquid ethanol within a container, where it can be rapidly cooled, wouldalso help introduction and dispersion while preventing potential thermalinjuries to sensitive commodities.

For example, with one embodiment, disinfestation of fruit flies from acommodity can be accomplished using 10 pressure cycles over a period ofan hour with a minimum pressure of approximately −10 psi and a maximumpressure of approximately +1 psi. In this embodiment, the ethanolfilling cycle starts with a minimum pressure of −14 psi and adding a 1psi vapor partial pressure of ethanol. Between pressure cycles, thecontainer is maintained at approximately −12.5 psi.

Ethanol influences the function of the central nervous system in man andit is likely that a similar activity occurs in insects and mites as theuse of ethanol combined with anoxia and CO₂ has been shown to be highlyeffective in controlling all developmental phases in various insects andmites. In addition, tests have shown that there are no detectablesensory effects on commodities such as table grapes, blueberries,raspberries, blackberries, oranges, and lemons with the use of ethanolto provide a secondary effect on the metabolism of pests.

Other materials that have been shown to be toxic or metabolic stimulantsto pests that are preferably in gas or vapor form may be introduced atblock 160 of FIG. 1. For example, the nitrogen oxides and the sulfuroxides have been used effectively. It is preferred that such toxics ormetabolic stimulants do not leave a residue if the commodity is forhuman consumption.

The use of regulated pesticides and fumigants may also be used in somecases. It will be seen that traditional toxic fumigants used at block160 can be administered in smaller concentrations in combination withthe anoxic environment than are customarily required to achieve toxicityalone. The quantity and time of exposure can also be adjusted to limitthe appearance of unwanted residues in the commodity.

Alternatively, in one embodiment, oxygen radicals and ozone can beproduced in situ within the commodity container preferably usingoscillating or pulsed radio frequency fields at block 170 of FIG. 1. Onesource of pulsed radio frequency fields is found in the apparatusdisclosed in U.S. Pat. No. 6,638,475 incorporated by reference herein.

Ozone (O₃) is a powerful oxidizing agent that leaves no toxic residueand can be added or generated in situ to further induce toxic effects tocellular metabolism in insects and mites. In situ generation also allowsthe use of the atomic oxygen precursor of molecular ozone as theinitiator of the oxidizing effects of O₃. Atomic oxygen, an extremelyrapid and reactive radical, is hundreds to thousands of times morereactive than molecular ozone.

Ozone may be formed near surfaces in air voids subjected to anoscillating electrical field being generated by pulsed RF power thatprovides a high probability for a direct effect on insects, mites, or onmicrobial contaminants present on the surface. An electric fieldpotential in air of between approximately 3 kV per centimeter toapproximately 5 kV per centimeter is usually required to produce ozoneat standard temperature and pressure.

In addition, the use of a low oxygen/high ballast gas environment incombination with the use of ozone has a synergistic effect. Therefore,the concentration of molecular ozone that is normally required fordisinfestation or disinfection may also be significantly reduced thusavoiding any secondary deleterious effect that may occur due to highermolecular ozone interactions with the host commodity such as oxidationor browning. The combination of the anoxic conditions with ozone (insitu and/or externally produced) allows the disinfestation process to becompleted with greater efficiency and in shorter times. Furthermore,using the known disinfection effects of ozone allows disinfestation anddisinfection effects to be simultaneously administered.

In addition to removing oxygen and creating a toxic environment, thisembodiment of the process may also increase the oxygen demand in insectsby applying an oscillating radiofrequency-generated electric field thatuses the conductivity of insects and mites to induce polarizationeffects. The oscillating electric field from a pulsed RF source forcesthe conductive insect and mites to physically react to the changingfield and induces small electric currents within the insects & mites,which results in the formation of dipoles. Polarized insects and mitesare forced to react by trying to orient themselves to the changingelectric field thus forcing them to increase their activity andaccelerate respiratory demands. Accordingly, radio frequency fields canbe applied to increase oxygen demands as well as generate reactiveoxygen radical or ozone gases.

The enhancement of oxygen demands in insects and mites can also beachieved with increased temperatures above room temperature sinceincreased temperatures force insects and mites to accelerate theirmetabolism. Increased temperatures may be limited in use totemperature-resistant commodities.

It can be seen that the process has multiple parameters that can betailored to pests and commodities with a wide range of characteristics.The secondary stress effects that are produced at block 130 usingoscillating electrical fields at block 170, disinfectants at block 140,antiseptics at block 150 or other toxics at block 160 can be conductedalone or in combination with the application of one or more of the othersecondary stressors. Simple manipulations of the parameters will allowthe process to be optimized to particular pests and host commodities andthe costs of administering the process can be minimized.

At the end of the treatment period, the commodities are removed from theanoxic environment of the container. The commodities may be treated inthe original bins or containers from the field or repackaged aftertreatment. At block 180 of FIG. 1, the treated commodities are stored orshipped to market. It is preferred that only treated commodities bestored together to avoid re-infestation.

Turning now to FIG. 2, a block diagram of one embodiment of theinvention 200 directed to disinfection of commodities from bacterial,fungal, protozoan or other microorganism is shown. The control ofmicroorganisms is one of the major concerns and challenges in foodproduction. The presence of microorganisms on a commodity can result inreduced shelf life and cross contamination during transportation andstorage of the host commodity. Controls are needed to retard or preventspoilage and to reduce or eliminate health hazards associated with foodsdue to microorganisms. The method 200 can be used for disinfection aloneor as part of a disinfestation and disinfection scheme. The method 200can also be used alone or in conjunction with traditional washing andother disinfection techniques that are presently used in food commodityprocessing.

At block 210 of FIG. 2, the commodity is sequestered in a preferably gastight container or enclosure. Agricultural commodities can be processedin containers from the field or may be transferred to containers beforeprocessing. The container or enclosure may be sized to accommodate anysize or shape of commodity packaging from individual packages topallets.

The sequestered commodities are exposed to a modified environmentcreated in the container preferably having extremely low oxygenconcentrations and high carbon dioxide concentrations for a period oftime determined by the type microorganism and the type of commodity atblock 220 of FIG. 2. The time of exposure typically ranges from a fewminutes to a few hours. The time of exposure can be lengthened ifnecessary with commodities such as nuts, grains or historical artifactsand the like that are not substantially effected by high concentrationsof carbon dioxide.

The preferred oxygen concentrations in the environment of the containerare preferably less than approximately 0.1% and carbon dioxideconcentrations greater than approximately 99.9%. Oxygen concentrationsof less than 0.01% are particularly preferred. Although carbon dioxideis preferred, nitrogen gas or a combination of carbon dioxide andnitrogen gas or other preferably inert ballast gas can be used.

The environment in the container or enclosure is preferably establishedby using pressure differentials that are applied with a combination ofvacuum and purging cycles. Once established, the environment ispreferably maintained at or near the atmospheric pressure of thesurroundings. The size of the pressure differentials should account forthe sensitivity of the commodity to positive or negative atmosphericpressures. The preferred range of pressures is approximately −20 psi toapproximately +5 psi. For pressure sensitive commodities, a range ofapproximately −10 psi to approximately +1 psi is preferred and used inshort periods of time in the order of tens of seconds to approximatelyone minute.

The method described herein combines the benefits of a low oxygen and ahigh carbon dioxide environment to affect the growth of both aerobic andanaerobic organisms. This is because the process involves a series ofenvironmental manipulations to rapidly change the chemical environmentwithin cells by depriving them of oxygen as well as affecting theacid-base balance and thus forcing an additional toxic effect createdwith a high CO₂ concentration purging the cellular gaseous environment.

As with insects, oxygen is an essential metabolite for the growth ofaerobic microorganisms. Depriving cells of the needed oxygen inducesmetabolic stress and death. The lack of oxygen deprives cells fromprocessing nutrients through oxidation-reduction reactions that areresponsible for energy production and synthesis. The elimination ofcarbon dioxide, an end product of respiration, is also critical tomaintaining cell viability and the acid-base balance at neutral pH (˜7)required for some microorganisms to survive and grow. However,microorganisms vary widely to their tolerance to carbon dioxide. In aCO₂ atmosphere, the growth of some organisms may be suppressed, whileothers may be less affected.

The created environment with excess CO₂ forces pH changes and increasesthe water activity required for cells to maintain vital metabolicfunctions. For example, carbon dioxide reacts with water to formcarbonic acid (H₂CO₃). Carbonic acid reacts chemically with basesforming bicarbonate (HCO₃ ⁻), which is the most common chemical form forcells to store carbon dioxide. Normal pH (pH ˜7) is regulated with thehydrated form of CO₂, that is carbonic acid (H₂CO₃), which in turn israpidly converted to bicarbonate (HCO₃ ⁻). Bicarbonate bufferingcapacity helps provide and maintain a neutral pH balance in cells.Therefore, as is done in this process, a rapid shift of the acid-basebalance is created in cells when exposed to high concentrations ofCO_(2.)This shift forces a lower pH (near pH 4) in cells causing stressand death to the cells or tissues.

It has been seen that the distribution of gaseous and dissolved carbondioxide flows from higher to lower pressures (or concentrations). As CO₂is formed within cells, the intracellular carbon dioxide will normallyflow to the cell's peripheral spaces where it is expelled through theair transport mechanisms. High concentrations of extra cellular carbondioxide lead to higher intracellular concentrations of carbon dioxidebyproducts. A forced high concentration of intracellular CO₂ alsoresults in deleterious effects on cellular metabolism most likely due tothe shift of the normal acid-base balance within cells to a more acidicpH range. It also increases the presence or retention of toxic CO₂ inthe cell while reducing its elimination.

Although some microorganisms may be expected to be more resistant tothese combined effects than insects, most microorganisms are affectedwith lower oxygen and high CO₂ environment. Even with relatively shortperiods of exposure, a reduction of the growth rate of largeconcentrations (˜10⁷ cfu/mL) of exogenous spoilage organisms (i.e.Botrytis sp., Penicillium sp., Phytophthora sp., Alternaria sp. Rhizopussp., etc.) as well as pathogenic bacteria, including Salmonellathyphimurium, Escherichia coli O157 H7, and Staphylococcus aureus havebeen demonstrated simultaneously with the disinfestation effectsdescribed herein.

To increase the mortality rate or extend the growth rate delays offungal organisms, bacteria and other microorganisms, the commodities areoptionally treated with disinfectants, antiseptics or other materialsthat are toxic to microorganisms or shown to increase the effect of theanoxic environment at block 230 of FIG. 2. The materials that areselected for use at block 230 preferably do not leave a residue on thecommodity that is toxic to humans and can be distributed in gas, vaporor aerosol form to the sequestered commodities. The materials that areselected for use can be directed at a particular microorganism and thesensitivity of a particular commodity to the material, if any.

A reduced pressure modified low oxygen environment is preferably used tocreate and distribute volatile chemicals with known disinfectionproperties at block 240, or known antiseptic properties at block 250 orother toxic properties at block 260 of FIG. 2. Although a reducedpressure environment is preferred, the secondary effects at block 230can be achieved with the introduction of disinfectants 240, antiseptics250 or other toxics 260 at a neutral or positive pressure in thecontainer. It will also be understood that more than one disinfectantcan be added to the anoxic environment sequentially or simultaneously atblock 240 of FIG. 2. Similarly, a disinfectant and an antiseptic ortoxic material can be added to the anoxic environment alone or incombination to achieve the desired effect.

One of the natural, short lived or easily removed chemicals that can beused as a disinfectant at block 240 of FIG. 2, is ozone (O₃) added fromexternal sources or generated in situ from its atomic (radical) oxygenprecursor (O.) using RF techniques. Ozone is a powerful oxidizing agentthat has a direct biocidal and biostatic effect on microorganisms. Theamount of ozone that is normally required to disinfect surfaces can begreatly reduced due to the synergistic effect of the anoxic atmosphereand the ozone on microorganisms.

In addition, low-level concentrations of hydrogen peroxide H₂O₂ (<1 g/L)of (10-30% v/v; m.p. −0.9° C.; p_(p)=1 mm at 15° C.) have been usedalone and in combination with other antiseptics to eliminate or suppressmicrobial growth. Hydrogen peroxide is useful because of its volatilityin low-pressure conditions as well as its disinfection capabilities.Although ozone and hydrogen peroxide are preferred disinfectants, otherantiseptics, including ethylene oxide and propylene oxide, may be usedalone or in combination with other materials.

Likewise, antiseptics such as ethanol or other alcohols are effective atcontrolling plant pathogens such as fungi, protozoa and bacteria.Antiseptics can be used alone or in combination with other antiseptics,disinfectants or toxics at block 250 of FIG. 2 to bolster the biocidaland biostatic effect of the anoxic environment that is created at block220.

One particularly useful combination of volatile chemicals isapproximately 30% hydrogen peroxide and approximately 70% ethanol byvolume introduced to the anoxic environment produced at block 220 ofFIG. 2. This combination is beneficial for both disinfection anddisinfestation because of the disinfectant characteristics as well asthe deleterious effects on the metabolism of insects and mites.

The method at block 260 of FIG. 2 also allows for the use of otherchemicals known for their rapid effects on microorganisms, singly or incombination. For example, gases of the group of nitrogen oxides or ofsulfur oxides, which have been used in some food processingapplications, may be used in addition to the anoxic environment. Sulfurdioxide (SO₂) for example, is a fumigant that is particularly useful inthe control of fungal organisms. However, its use may be limited byexisting regulatory limits for residues in foods (i.e. <10 ppm) due tothe fact that it induces the formation of sulfites, a chemical known tohave adverse effects in certain sectors of the human population. Sulfurdioxide is also used extensively in the food industry as a bleachingagent.

Other regulated and non-regulated bactericides and fungicides may alsobe used on commodities at block 260 of FIG. 2 depending on the nature ofthe commodity and the types of microorganisms that are prevalent. Thecombination of the low oxygen and high carbon dioxide environment andsecondary bactericides or fungicides allows smaller quantities ofsecondary materials to be used to achieve the desired results.Consequently, the volume of bactericide or fungicide that is applied maybe substantially less than is required to kill or delay the growth ofpests when used alone. Therefore, the residues that may be present atblock 260 will be substantially smaller and within acceptable limits forhuman or animal consumption.

Accordingly, the biocidal or biostatic effect on microorganisms of thelow oxygen-high carbon dioxide or other ballast gas environment createdat block 220 can be enhanced with the use of volatile disinfectants 240or antiseptics 250 such as ozone, ethanol, or hydrogen peroxide usedsingly or in combination. The use of these materials may also reduce thetime of exposure of the commodity to the anoxic environment as well asthe overall processing time for disinfection within the range of severalseconds to minutes.

It can also be seen that the effect of a low oxygen and high carbondioxide environment contributes and combines to induce rapid mortalityin insects while simultaneously causing metabolic biocidal or biostaticeffects on microorganisms. Since exogenous fungal and pathogenicorganisms are usually present in perishable food commodities,microorganisms can also be treated along with insects by the controlledenvironment as oxygen and carbon dioxide are critically related to theircellular metabolism and survival.

As a result of the process, the rate of microbial growth is eradicatedor decreased significantly allowing for a longer shelf life of thecommodity, which can be further extended under refrigerated storage.Disinfection effects have also been shown in injured fruit leading tobiocidal and/or biostatic effects.

After exposure to the controlled environment for a sufficient period oftime, the commodities are removed from the closed container at block 270of FIG. 2 and prepared for shipping or storage. Traditional handling,storage and shipping of treated commodities can be used if care is takento avoid re-infection.

The invention may be better understood with reference to theaccompanying examples, which are intended for purposes of illustrationonly and should not be construed in any sense as limiting the scope ofthe present invention as defined in the claims appended hereto.

Samples of insects and mites including various types of Thrips(Frank-liniella occidentalis), Fruit Flies (Drosophila melanogaster),Ants (Pogonomyrmex subdentata), Aphids (Myzus persicae), Harlequin Bugs(Murgantia histrionica), and Mites (Amblyseius cucumeris, Tetranychusurticae) were used to demonstrate the disinfestation capabilities of low(hypobaric) or high (hyperbaric) pressure low oxygen and high carbondioxide concentration environments on a variety of insect pests. Theaddition of antiseptics, disinfectants and radio frequency (RF) andozone to the anoxic environment was also used on infestations of mitesand other pests. Treated and non-treated (control) samples for someinsects included all mobile life cycles (i.e. juvenile, adults, pupas,larva, etc.) as well as eggs.

Example 1

Experiments involving a hypobaric condition were conducted with aPrecision Vacuum Oven (PVO) provided with a manometer (−30 inches of Hgto +15 psi range) and internal Hg thermometer and were modified to allowfor vacuum and pressurized purging operations with different gasmixtures. A dry pump was used to establish vacuum up to −14 psi (−724 mmHg). Purging was conducted with carbon dioxide up to reaching barometricpressures or slightly below (−1 psi). Carbon Dioxide was used as asource for purging and for establishing the high concentration carbondioxide environments. Nitrogen gas was also used as an inert ballast gasto utilize different CO₂ gas environments to establish the properconditions in some experiments.

The effect of a low oxygen/high carbon dioxide environment at below theambient barometric pressure on Ants, Mites, Thrips, Harlequin Bugs,Aphids, Fruit Flies at different stages of development was evaluated. Itcan be seen that the controlling effect of an anoxic hypobaric carbondioxide environment on mortality of the subject insects is significant.Observations of the hypobaric treatments were made immediately after thetreatment and mortality was observed 24 hours after the treatment.

Control of adult Ants in a hypobaric environment with at least onepressure cycle is found in Table 1. In one experiment the Ants weretotally immobilized and eradicated with an 8-hour treatment.

Significant mortality in adult and juvenile mites as well as Mite eggswith hypobaric treatments was seen in the results in Table 2 throughTable 5. Sizeable pressure changes alone in a low oxygen atmosphere werenot as effective as minor pressure changes in a low oxygen high carbondioxide environment. Greater than 90 percent mortality for adult andjuvenile Mites was observed with treatment times in the range of 4 to 7hours with a pressure differential of −2 psi.

Adult Harlequin Bugs and Aphids are particularly susceptible to thehypobaric method, as total mortality was seen after seven hours oftreatment as shown in Table 6 and Table 7 respectively.

Fruit fly adults had significant mortality 24 hours after treatment asseen in Table 8 with treatment times from 7 to 9 hours. Short durationpressure changes with a minor pressure differential were seen to beeffective.

Tables 9 through 11 show the effect of the hypobaric treatments on Thripadults, pupas and eggs. Eight-hour treatment times were seen to beeffective for adult and pupa thrips but only marginally effective on theviability of Thrip eggs.

Example 2

To demonstrate the effectiveness of the procedure in a hyperbaricenvironment, experiments were conducted with a pressure chamber (PC)fitted with a manometer (0 to +15 psi) and proper on/off valves. Thechamber was capable of withstanding 5-10 psi pressure without major gaslosses for over a 24-hour period.

Purging was conducted in multiple cycles with a high-pressure carbondioxide flow directly from 2,500-psi storage cylinders at roomtemperature. Pressure (+2 to +5 psi) was determined with manometers andprovided a rapid purge of the pressure chamber environment containingthe samples to be treated. During purging, a carbon dioxide/Air volumeratio greater than 3 was used to assure proper air removal.

Table 12, shows the effects of the treatment at hyperbaric pressures onadult ants with three short duration hyperbaric pressure cycles. TheAnts show a greater resistance to the hyperbaric anoxic conditions thatto the hypobaric treatments.

Control of different species of Mites at various stages of developmentfrom egg to adult to cycled hyperbaric anoxic conditions is shown inTables 13 through Table 17. The results illustrate significant mortalityafter 24 hours in adult and juvenile mites that have had 6 to 7 hourexposures with a small number of short duration pressure cycles. Miteeggs had 80% mortality after a 6-hour exposure and it is expected thatlonger exposure times will increase mortality of mite eggs.

Similarly, total mortality was seen in Harlequin Bugs and Aphids asshown in Table 18 and Table 19 respectively. Total mortality was shownin 7 and 8-hour exposures with three short duration hyperbaric pressurecycles.

Table 20 shows the control of fruit fly adults after a 7 to 9 hourexposure to the hyperbaric treatment environment. Greater than 90%mortality can be achieved with three short duration hyperbaric pressurecycles.

The control of Thrips in egg, pupae and adult stages are shown in Tables21-23. Greater than 90% mortality was observed in adults, pupas and eggswith five short duration pressure cycles.

It can be seen that a wide variety of insects and mites in various formscan be eliminated with the cycling of hyperbaric or hypobaric pressuresof a low oxygen/high carbon dioxide environment. Time of exposure to theenvironment is less than 24 hours and typically less than 8 hours.

Example 3

A demonstration of the effect of a low oxygen-high ballast gasenvironment with a pressure differential with cycles of below barometricpressure to above barometric pressure and a metabolic stressor on fruitflies was conducted for a short treatment time of one hour. Thedeleterious effects of hypobaric and hyperbaric conditions with ananoxic environment and the presence of ethanol to provide additionalmetabolic stress were observed with fruit flies at all biologicalstages. The various biological forms of fruit flies were exposed to alow oxygen-high carbon dioxide environment with 10 cycles of a pressuredifferential ranging from −20 inches of Hg to +2 inches of Hg for a onehour treatment period. Ethanol vapor at 1.3 to 2.0 in Hg vapor pressurewas introduced to the environment as a metabolic stressor. The resultsare shown in Table 24. It can be seen that adult, pupa, larval and eggforms of fruit flies were completely eradicated with a one hourtreatment period. The survival rate for eggs was determined by enclosionrates at beyond 24 hours.

Example 4

The effect of secondary metabolic stress mechanisms on pests in ahyperbaric anoxic environment was demonstrated on the juvenile and adultmites. Experiments with pulsed RF fields were conducted with alaboratory-scale system fitted with a parallel-plate capacitor poweredwith 15 kV NC external transformer. Oscillating electric fields with >5kV/cm were thus utilized in these combined experiments.

Table 25 illustrates the control of juvenile and adult mites with thesecondary stress exerted by a 60 Hz frequency. It can be seen that theaddition of a secondary stressor increases the mortality of juvenile andadult mites over the anoxia treatment alone.

Example 5

The synergistic effect of pulsed RF fields and a transient toxic gassuch as molecular ozone to the eggs as well as juvenile and adult mitesis demonstrated in the combined results shown in Table 26. Experimentswith atomic oxygen and/or molecular ozone were conducted in a separatelaboratory-scale system also fitted with a parallel-plate capacitorpowered with a 15 kV A/C RMS external transformer. In these experiments,in situ production of atomic oxygen and molecular ozone took placewithin the parallel-plate capacitor with oscillating electric fields >5kV/cm. Parallel, comparison experiments involving molecular ozone wereconducted with an external ozone generator feeding the parallel-platecapacitor system thus combining RF and Ozone interactions.

It can be seen that the combination of oscillating electric fields toincrease oxygen demand and toxic gases such as atomic oxygen or ozonewithin a cycled carbon dioxide anoxic environment results in essentiallytotal mortality in the eggs, juvenile and adult forms of Mites.

Example 6

Microbial growth rates (i.e. inhibition or retardation) at roomtemperature have been observed in several molds and bacteria pathogenswhen exposed to the anoxic conditions (i.e. hyperbaric or hypobaricanoxia with high carbon dioxide environment) as provided for insectdisinfestation. In order to document these effects, experiments werecarried out with Botrytis cinerea, Penicillium italicum, Alternariaalternata, Salmonella thyphimurium, and Escherichia coli O157:H7.

Inoculum concentrations ranging from 10² to 10⁷ cfu/mL were plated ontoculture plates containing an appropriate growth media, dried in alaminar flow hood, and exposed to the anoxic/carbon dioxide environment.After completion of the exposure time to the regulated gaseousenvironment, the plates were removed and placed on closed containers forincubation at room temperature (˜22° C.). Observations and assays ofmicrobial populations were conducted periodically over subsequent days.

Microbial assays for Botrytis cinerea (10³ cfu/mL) treated with cycledanoxic/high carbon dioxide environs for 16 hours at hyperbaric andhypobaric pressures were conducted. Initial growth in treated platesstarted approximately 2-2.5 days after treatment while growth in thecontrol plates was immediate. It was observed that the hyperbaricprocess was more effective in causing the growth inhibition effect thanthe hypobaric process under the conditions tested. Colony countingindicated approximately a 300 times lower population in treated samplesover the control samples.

Microbial assays for Penicillium italicum (10⁷ cfu/mL) treated withanoxic/high carbon dioxide environment according to the invention for 16hours at hyperbaric and hypobaric pressures were also conducted. Initialgrowth in treated plates started approximately 1-1.5 days aftertreatment while growth in the control plates was immediate. Colonycounting at this time indicated an approximately 1,000 times lowerpopulation in treated samples, with the hyperbaric process beingapproximately 10 times better than the effects with the hypobaricprocess.

Assays for Alternaria alternata (10⁶ cfu/mL) treated with anoxic/highcarbon dioxide environments for 16 hours at hyperbaric and hypobaricpressures were also conducted. No growth in treated plates was observedafter approximately 3-3.5 days after treatment.

Microbial assays for Salmonella thyphimurium (10² cfu/mL) treated withanoxic/high carbon dioxide environments for 16 hours at hyperbaric andhypobaric pressures were also conducted. No growth was observed in thetreated plates one day after processing. At the onset of growth, colonycounting indicated that there were no differences in the number ofcolonies. However, a significant difference in the stage of developmentof the colonies compared to the control was observed.

Example 7

The addition of low concentrations of a vaporized disinfectant to theanoxic/high carbon dioxide environment has been shown to effectivelyeliminate some fungi and bacteria with the methods of the presentinvention. The biocidal effect at room temperature of the method wasdemonstrated with several fungi and bacteria pathogens exposed to ananoxic & ethanol environment using identical processing as fordisinfestation described previously. Experiments were carried out withplant pathogens such as Alternaria alternata, Rhizopus sp., andPenicillium sp, as well as with human pathogens such as Salmonella sp.,Escherichia coli O157:H7, and Staphylococcus aureus.

Generally, inoculum concentrations ranging from 10⁴ to 10⁵ cfu/mL wereplated onto culture plates containing an appropriate growth media, driedin a laminar flow hood, and exposed to the anoxia & ethanolenvironments. After completion of the exposure time to the regulatedgaseous environment, the culture plates were removed and placed onclosed containers for incubation at room temperature (˜22° C.).Observations and assays of microbial populations were conductedperiodically over subsequent days.

Microbial assays for Alternaria alternata (10⁴ cfu/mL) treated with anenvironment of anoxic/high carbon dioxide and low concentrations of 70%vaporized ethanol and 30% of vaporized hydrogen peroxide for 6 hours didnot produce any growth in treated plates when observed approximately 6days after treatment. The control plates were essentially covered withgrowth after 6 days.

Microbial assays of Rhizopus sp. (10⁴ cfu/mL) treated with anenvironment of anoxic/high carbon dioxide and low concentrations of 70%vaporized ethanol and 30% of vaporized hydrogen peroxide for 6 hourswere conducted. After 6 days, no growth was observed in the treatedplates and the control plates were virtually covered with growth.

Similarly, microbial assays of Penicillium sp (10⁴ cfu/mL) treated withan environment of anoxic/high carbon dioxide and low concentrations of70% vaporized ethanol and 30% of vaporized hydrogen peroxide for 6 hoursdid not produce any growth in the treated plates while the controlplates were covered after 6 days.

Microbial assays of Salmonella sp. (10⁵ cfu/mL) treated with anenvironment of anoxic/high carbon dioxide and low concentrations of 70%vaporized ethanol and 30% of vaporized hydrogen peroxide for 6 hourswere conducted. After 4 days, no growth was observed in the treatedplates and the control plates exhibited substantial growth.

Likewise, Microbial assays of Escherichia coli O157:H7 (10⁵ cfu/mL) andStaphylococcus aureus (10⁵ cfu/mL) treated with an environment ofanoxic/high carbon dioxide and low concentrations of 70% vaporizedethanol and 30% of vaporized hydrogen peroxide for 6 hours produced nogrowth.

Example 8

The capability of the method for disinfection of the surfaces of severalfresh fruits was evaluated. Raspberries, blackberries, table grapes, andstrawberries were selected since these fruits are often infected withnatural flora and can spoil rapidly within a few days if kept at roomtemperature. Healthy, non-injured fruit was chosen from batches ofcommercial quality fruit. Control and treated samples from the samebatch were kept at room temperature (˜22° C.) in sterile dishes and at alaminar flow hood without ventilation. Daily observations were made forthe onset and propagation of infective sites.

Raspberries were treated with an environment of anoxic/high carbondioxide and low concentrations of 70% vaporized ethanol. The time oftreatment was 48 hours. After 11 days the control fruit was fullyengulfed with natural flora while the treated berries showed no sign ofgrowth.

Blackberries and strawberries were also treated with an environment ofanoxic/high carbon dioxide and low concentrations of 70% vaporizedethanol with a treatment time of 48 hours and the treated fruit showedno sign of growth of natural flora when observed 11 days aftertreatment. The control fruit showed significant growth of natural flora.

Finally, table grapes were also treated with an environment ofanoxic/high carbon dioxide for 16 hours and low concentrations of 70%vaporized ethanol for 2 hours. After 7 days at room temperature thecontrol grapes were covered with a substantial growth of natural florawhile the treated grapes showed no signs of growth.

Example 9

The potential of the method to disinfect injured fruit tissues andaffect the development of microbial inoculums was evaluated. Postharvest infections through injury are believed to be the cause of alarge fraction of spoilage losses in agriculture.

As discussed previously, one embodiment of the present methods providesa reduced pressure environment particularly suited for expanding orvolatilizing some chemical disinfectants with relatively high partialpressures such as ethanol or hydrogen peroxide or to introduce othergases or pesticides into the environment of the container. The testswere conducted with inoculated and injured fruits that were latersubjected to the cycled low oxygen/high carbon dioxide environment.

The biostatic effect of the method on Penicillium digitatum in freshValencia oranges was shown. Oranges were punctured (1×3 mm deep) duringinoculation (10⁶ cfu/mL) thereby allowing the inoculation to reachlayers of tissue under the skin. The injury remained partially openduring the treatment. The treated oranges were treated with anenvironment of anoxic/high carbon dioxide for 45 minutes. The treatedand control oranges were stored at room temperature. The control orangesexhibited substantial growth of Penicillium digitatum within six days ofinoculation. No growth was observed on the treated oranges. Resultsindicated that the treatment method is capable of either reducingmicrobial growth (a biostatic effect) or causing disinfection (biocidaleffects) to both plant and human pathogens.

Accordingly, it will be seen that this invention provides an effectiveand efficient apparatus and method for disinfesting commodities ofinsects, mites and other organisms and is a viable alternative to theuse of methyl bromide fumigation. It will also be seen that theinventions provides an effective apparatus and method for disinfectingcommodities from bacterial, fungal and other microbial pests.

Although the description above contains many details, these should notbe construed as limiting the scope of the invention but as merelyproviding illustrations of some of the presently preferred embodimentsof this invention. Therefore, it will be appreciated that the scope ofthe present invention fully encompasses other embodiments which maybecome obvious to those skilled in the art, and that the scope of thepresent invention is accordingly to be limited by nothing other than theappended claims, in which reference to an element in the singular is notintended to mean “one and only one” unless explicitly so stated, butrather “one or more.” All structural, chemical, and functionalequivalents to the elements of the above-described preferred embodimentthat are known to those of ordinary skill in the art are expresslyincorporated herein by reference and are intended to be encompassed bythe present claims. Moreover, it is not necessary for a device or methodto address each and every problem sought to be solved by the presentinvention, for it to be encompassed by the present claims. Furthermore,no element, component, or method step in the present disclosure isintended to be dedicated to the public regardless of whether theelement, component, or method step is explicitly recited in the claims.No claim element herein is to be construed under the provisions of 35U.S.C. 112, sixth paragraph, unless the element is expressly recitedusing the phrase “means for.

TABLE 1 Control of Ants in a Hypobaric Anoxic/High Carbon Dioxide (−2psi) Environment Treatment Effect Time Parameter After Mortality (hours)(Hypobaric) Treatment 24 h later 15 h under vacuum 100% >80% (−28 psi)immobilized 17 h CO₂ (−2 psi)  90% >90% immobilized 19 h CO₂ (−2 psi) 90% 100% immobilized 7 h CO₂ (−2 psi)  90% >90% immobilized 15 h CO₂(−2 psi) 100% >80% immobilized 8 h CO₂ (−2 psi) 100% 100% immobilized

TABLE 2 Control of “Thripex” Mites (Amblyseius cucumeris) in a HypobaricAnoxic/High Carbon Dioxide (−2 psi) Environment Treatment Effect TimeParameter After Mortality (hours) (Hypobaric) Treatment 24 h later 15 hvacuum >50% >50% (−28 psi) immobilized 4.5 h CO₂ (−2 psi) >90% >95%immobilized 17 h CO₂ (−2 psi) >80% >95% immobilized 19 h CO₂ (−2psi) >90% >98% immobilized 7 h CO₂ (−2 psi) >90% >99% immobilized 7 hCO₂ (−2 psi) 100% >90% immobilized 15 h CO₂ (−2 psi) 100% >98%immobilized 6.5 h CO₂ (−2 psi) 100% >95% immobilized

TABLE 3 Control of Adult Mites (Tetranychus urticae) with a HypobaricAnoxic/High Carbon Dioxide (−2 psi) Environment Time Treatment HypobaricAfter Mortality (hours) Parameter Cycles Treatment 24 h later 8 h CO₂ 4min. 5 purge 100% 95% w/CO₂ immobilized (95/100) 8 h CO₂ 4 min. 5 purge100% 94% w/CO₂ immobilized (94/100)

TABLE 4 Control of Juvenile Mites (Tetranychus urticae) with a HypobaricAnoxic/High Carbon Dioxide (−2 psi) Environment Time Treatment HypobaricAfter Mortality (hours) Parameter Cycles Treatment 24 h later 6 h CO₂ 4min. 5 purge 100% 90% w/CO₂ immobilized (180/200) 6 h CO₂ 4 min. 5 purge100% 90% w/CO₂ immobilized (180/200)

TABLE 5 Control of Mite Eggs (Tetranychus urticae) with a HypobaricAnoxic/High Carbon Dioxide (−2 psi) Environment Time Treatment HypobaricAfter Mortality (hours) Parameter Cycles Treatment 24 h later 6 h CO₂ 4min. 5 purge N/A >80% w/CO₂ 6 h CO₂ 4 min. 5 purge N/A >90% w/CO₂

TABLE 6 Control of “Harlequin Bugs” (Murgantia histrionica) in aHypobaric Anoxic/High Carbon Dioxide (−2 psi) Environment TreatmentEffect Time Parameter After Mortality (hours) (Hypobaric) Treatment 24 hlater 18 h CO₂ (−2 psi)  80% 100% immobilized 6 h CO₂ (−2 psi)  80%  80%immobilized 18 h CO₂ (−2 psi) 100% 100% immobilized 7 h CO₂ (−2 psi)100% 100% immobilized 8 h with CO2 100% 100% (−2 psi) immobilized

TABLE 7 Control of Various Aphids in a Hypobaric Anoxic/High CarbonDioxide (−2 psi) Environment Treatment Effect Time Parameter AfterMortality (hours) (Hypobaric) Treatment 24 h later 6 h CO₂ (−2 psi) 100%100% immobilized 19 h  CO₂ (−2 psi) 100% 100% immobilized 7 h CO₂ (−2psi) 100% 100% immobilized 7 h CO₂ (−2 psi) 100% 100% immobilized 8 hCO₂ (−2 psi) 100% 100% immobilized

TABLE 8 Control of Adult Fruit Flies (Drosophila melanogaster) with aHypobaric Anoxic/High Carbon Dioxide (−2 psi) Environment Time TreatmentHypobaric After Mortality (hours) Parameter Cycles Treatment 24 h later7 h CO₂ 1 min. 3 purge 100% 76% with CO₂ immobilized (53/70) 8 h CO₂ 1min. 3 purge 100% 90% with CO₂ immobilized (73/80) 9 h CO₂ 1 min. 3purge 100% 94% with CO₂ immobilized (75/80) 9 h CO₂ 1 min. 3 purge 100%90% with CO₂ immobilized (45/50)

TABLE 9 Control of Adult Thrips (Frankliniella occidentalis) with aHypobaric Anoxic/High Carbon Dioxide (−2 psi) Environment Time TreatmentHypobaric After Mortality (hours) Parameter Cycles Treatment 24 h later8 h CO₂ 3 min. 5 purge 100% 96% with CO₂ immobilized (192/200) 7 h CO₂ 3min. 5 purge 100% 80% with CO₂ immobilized (160/200)

TABLE 10 Control of Pupa Thrips (Frankliniella occidentalis) with aHypobaric Anoxic/High Carbon Dioxide (−2 psi) Environment Time TreatmentHypobaric After Mortality (hours) Parameter Cycles Treatment 24 h later8 h CO₂ 3 min. 5 purge 100% 90% with CO₂ immobilized (180/200)

TABLE 11 Control of Thrip Eggs (Frankliniella occidentalis) with aHypobaric Anoxic/High Carbon Dioxide (−2 psi) Environment Time TreatmentHypobaric After Mortality (hours) Parameter Cycles Treatment 24 h later8 h CO₂ 3 min. 5 purge N/A 50% with CO₂ (125/250)

TABLE 12 Control of Adult Ants (Pogonomyrmex subdentata) With aHyperbaric Anoxic/High Carbon Dioxide (+5 psi) Environment TimeTreatment Hyperbaric After Mortality (hours) Parameter Cycles Treatment24 h later  7 h CO₂ 3 pressurized, 100% 80% 1 min. immobilized (4/5) 15h CO₂ 3 pressurized, 100% 60% 1 min. immobilized (3/5)

TABLE 13 Control of Adult Mites (Tetranychus urticae) With a HyperbaricAnoxic/High Carbon Dioxide (+5 psi) Environment Time TreatmentHyperbaric After Mortality (hours) Parameter Cycles Treatment 24 h later6 h CO₂ 5 pressurized, 100% 99% 1 min. immobilized (198/200) 6 h CO₂ 5pressurized, 100% 93% 1 min. immobilized (107/115)

TABLE 14 Control of Adult Mites (Amblyseius cucumeris) With a HyperbaricAnoxic/High Carbon Dioxide (+5 psi) Environment Time TreatmentHyperbaric After Mortality (hours) Parameter Cycles Treatment 24 h later7 h CO₂ 3 pressurized, >90% 98% 1 min. immobilized  (98/100) 7 h CO₂ 3pressurized, >90% 90% 1 min. immobilized (180/200) 15 h  CO₂ 3pressurized, >98% 98% 1 min. immobilized (196/200)

TABLE 15 Control of Juvenile Mites (Tetranychus urticae) With aHyperbaric Anoxic/High Carbon Dioxide (+5 psi) Environment TreatmentHyperbaric After Mortality Time Parameter Cycles Treatment 24 h later 6h CO₂ 5 pressurized, 100% 90% 1 min. immobilized  (90/100) 6 h CO₂ 5pressurized, 100% 90% 1 min. immobilized (115/125)

TABLE 16 Control of Mite Eggs (Tetranychus urticae) With a HyperbaricAnoxic/High Carbon Dioxide (+5 psi) Environment Treatment HyperbaricAfter Mortality Time Parameter Cycles Treatment 24 h later 6 h CO₂ 5pressurized, N/A 80% 1 min. (240/300)

TABLE 17 Control of Adult Mites (Amblyseius cucumeris) With a HyperbaricAnoxic/High Carbon Dioxide (+5 psi) Environment Treatment HyperbaricAfter Mortality Time Parameter Cycles Treatment 24 h later 7 h CO₂ 3pressurized, >90% 98% 1 min. immobilized  (98/100) 7 h CO₂ 3pressurized, >90% 90% 1 min. immobilized (180/200) 15 h  CO₂ 3pressurized, >98% 98% 1 min. immobilized (196/200)

TABLE 18 Control of Adult Harlequin Bugs (Murgantia histrionica) With aHyperbaric Anoxic/High Carbon Dioxide (+5 psi) Environment TreatmentHyperbaric After Mortality Time Parameter Cycles Treatment 24 h later 19h  CO₂ 3 pressurized, 100% 100% 1 min. immobilized 7 h CO₂ 3pressurized, 100% 100% 1 min. immobilized 8 h CO₂ 3 pressurized, 100%100% 1 min. immobilized

TABLE 19 Control of Adult Aphids (Myzus persicae) And Adult Thrips(Frankliniella occidentalis) With a Hyperbaric Anoxic/High CarbonDioxide (+5 psi) Environment Treatment Hyperbaric After Mortality TimeParameter Cycles Treatment 24 h later 19 h  CO₂ 3 pressurized, 100% 100%1 min. immobilized (30/30) 7 h CO₂ 3 pressurized, 100% 100% 1 min.immobilized (30/30) 7 h CO₂ 3 pressurized,  50% 100% 1 min. immobilized(30/30) 8 h CO₂ 3 pressurized, 100% 100% 1 min. immobilized (30/30)

TABLE 20 Control Adult of Fruit Flies (Drosophila melanogaster) With aHyperbaric Anoxic/High Carbon Dioxide (+5 psi) Environment TreatmentHyperbaric After Mortality Time Parameter Cycles Treatment 24 h later 7h CO₂ 3 pressurized, 100% 80% 1 min. immobilized (48/60) 8 h CO₂ 3pressurized, 100% 90% 1 min. immobilized (73/80) 9 h CO₂ 3 pressurized,100% 94% 1 min. immobilized (75/80) 9 h CO₂ 3 pressurized, 100% 90% 1min. immobilized (45/50)

TABLE 21 Control of Adult Thrips (Frankliniella occidentalis) With aHyperbaric Anoxic/High Carbon Dioxide (+5 psi) Environment TreatmentHyperbaric After Mortality Time Parameter Cycles Treatment 24 h later 8h CO₂ 5 pressurized, 100% 90% 1 min. immobilized (180/200) 7 h CO₂ 5pressurized, 100% 95% 1 min. immobilized (190/200)

TABLE 22 Control of Thrip Pupas (Frankliniella occidentalis) With aHyperbaric Anoxic/High Carbon Dioxide (+5 psi) Environment TreatmentHyperbaric After Mortality Time Parameter Cycles Treatment 24 h later 8h CO₂ 5 pressurized, 100% 99% 1 min. immobilized (198/200)

TABLE 23 Control of Thrip Eggs (Frankliniella occidentalis) With aHyperbaric Anoxic/High Carbon Dioxide (+5 psi) Environment TreatmentHyperbaric After Mortality Time Parameter Cycles Treatment 24 h later 8h CO₂ 5 pressurized, N/A 90% 1 min. (150/167)

TABLE 24 Control of “Fruit Flies” (Drosophila melanogaster) in a cycledHyperbaric/Hypobaric (−20 Hg to +2 Hg) and an Anoxic/High Carbon Dioxideand Ethanol Vapor Environment Time Treatment Parameter Effect Mortality(hours) (Hypobaric) After Treatment 24 hrs later 1 h CO₂/10 cycles 100%Adult 100% (−20 to +2 psi) Mortality 1 h CO₂/10 cycles 100% Pupa 100%(−20 to +2 psi) Mortality 1 h CO₂/10 cycles 100% Larva 100% (−20 to +2psi) Mortality 1 h CO₂/10 cycles 100% Egg 100% (−20 to +2 psi) Mortality

TABLE 25 Control of Juvenile and Adult Mites (Amblyseius cucumeris) WithAnoxia & Radio Frequency Secondary Effects Treatment After MortalityTime Parameter Observations Treatment 24 h after 16 h With CO₂ & 3cycles,  99% 99% 60 Hz 1 min immobilized 16 h With CO₂ & 3 cycles, 100%80% 60 Hz 1 min immobilized 12 h With CO₂ & 30 cycles,  100% 100%  60 Hz1 min immobilized

TABLE 26 Control of Egg, Juvenile and Adult Mites (Amblyseius cucumeris)With The Use Of RF and Ozone Electrode to Treatment Commodity AfterMortality Date Parameter Distance Treatment 24 h after 7 h 60 Hz, O₃3.8-cm gap 100% 100% immobilized (911/911) 15.5 h 60 Hz, O₃ 3.8-cm gap 97% 100% immobilized (1002/1002) 16 h 60 Hz, O₃ 3.8-cm gap 100% 100%immobilized 7 h 60 Hz, O₃ 3.8-cm gap 100% 100% immobilized 16 h 60 Hz,O₃ 3.8-cm gap 100% 100% immobilized (1024/1024) 10 h 60 Hz, O₃ 3.8-cmgap 100% 97.5%  immobilized (390/400) 16 h 16 h, 3.8-cm gap 100% 99.5% 60 Hz, O₃ immobilized (656/659) 16 h 16 h, 3.8-cm gap 100%  90% 60 Hz,O₃ immobilized 10 h 10 h, 3.8-cm gap 100% 100% 60 Hz, O₃ immobilized(1393/1393) 16 h 16 h, 3.8-cm gap 100% 100% 60 Hz, O₃ immobilized(1646/1646) 10 h 10 h, 3.8-cm gap 100% 99.7%  60 Hz, O3 immobilized(820/822) 10 h 10 h, 3.8-cm gap 100% 99.5%  60 Hz, O₃ immobilized(1388/1395) 10 h 10 h, 3.8-cm gap 100%  99% 60 Hz, O₃ immobilized 15 h15 h, 3.8-cm gap 100% 100% 60 Hz, O₃ immobilized 17 h 17 h, 7.5-cm gap 97%  93% 60 Hz, O₃ immobilized (650/700) 4 h 4 h, 3.8-cm gap  99%  99%60 Hz, O₃ immobilized 10 h 10 h, 3.8-cm gap 100% 100% 60 Hz, O₃immobilized 16 h 16 h, 3.8-cm gap 100% 100% 60 Hz, O₃ immobilized

1-113. (canceled)
 114. A method for insect and mite control, comprising:depriving insect or mite pests of oxygen; introducing metabolic stressin said pests while in an oxygen deprived state by subjecting said peststo an oscillating radiofrequency field; exposing said pests to apressure differential environment, said pressure differential having amaximum pressure and a minimum pressure; and cycling the pressure of theenvironment from said minimum pressure to said maximum pressure and backfor a plurality of cycles.
 115. A method as recited in claim 114,wherein said maximum pressure and said minimum pressure of said pressuredifferential are at a level that is greater than the ambient barometricpressure.
 116. A method as recited in claim 114, wherein said maximumpressure and said minimum pressure of said pressure differential are ata level that is below the ambient barometric pressure.
 117. A method asrecited in claim 114, wherein said maximum pressure is at a level thatis greater than the ambient barometric pressure and said minimumpressure is at a level that is less than the ambient barometricpressure.
 118. A method as recited in claim 114, wherein said pressuredifferential comprises: a maximum pressure of approximately −2 poundsper square inch; and a minimum pressure of approximately −10 pounds persquare inch.
 119. A method as recited in claim 114, wherein saidpressure differential comprises: a maximum pressure of approximately +2pounds per square inch; and a minimum pressure of approximately +10pounds per square inch.
 120. A method as recited in claim 114, whereinsaid oscillating radiofrequency field is configured to produce ozone.121. A method as recited in claim 114, wherein said oscillatingradiofrequency field is configured to produce oxygen radicals.
 122. Amethod as recited in claim 114, wherein said oscillating radiofrequencyfield has a field strength of greater than approximately 3 KV/cm.
 123. Amethod as recited in claim 114, wherein said step of depriving an insector mite of oxygen comprises: subjecting said insect or mite pests to alow oxygen and a high ballast gas environment with approximately 0.01%to approximately 1% oxygen by volume.
 124. A method as recited in claim123, wherein said ballast gas comprises nitrogen gas, carbon dioxide gasor a combination of nitrogen gas and carbon dioxide gases.
 125. A methodas recited in claim 123, wherein said low oxygen and high ballast gasenvironment comprises less than approximately 0.1% oxygen and greaterthan a combination of approximately 49.9% nitrogen gas and approximately50% carbon dioxide gas by volume.
 126. A method as recited in claim 114,further comprising: exposing said insect or mite pests to volatilechemicals known to cause metabolic stress in insects or mites.
 127. Amethod as recited in claim 126, wherein said chemicals comprise achemical from the group of chemicals consisting essentially of analcohol, an insecticide, an oxygen radical, ozone, hydrogen peroxide,the nitrogen oxides group of compounds; and the sulfur oxides group ofcompounds.
 128. A method for controlling an insect, mite or otherbiological pest, comprising: exposing pests to a reduced oxygenenvironment for a period of time; manipulating the respiratory system ofsaid pest; exposing said pest to a radio frequency field for a period oftime; and exposing said pest to at least one chemical shown to causemetabolic stress in insects, mites and other biological pests.
 129. Amethod as recited in claim 128, wherein said manipulation of therespiratory system of a pest comprises: decreasing the pressure of thereduced oxygen environment to a level below the ambient barometricpressure; increasing the pressure of the reduced oxygen environment to alevel below the ambient barometric pressure; and repeating the cycle ofincreasing and decreasing environmental pressure multiple times.
 130. Amethod as recited in claim 128, wherein said manipulation of therespiratory system of a pest comprises: increasing the pressure of thereduced oxygen environment to a level above the ambient barometricpressure; decreasing the pressure of the reduced oxygen environment to alevel above the ambient barometric pressure; and repeating the cycle ofincreasing and decreasing environmental pressure multiple times.
 131. Amethod as recited in claim 128, wherein said manipulation of therespiratory system of a pest comprises: increasing the pressure of thereduced oxygen environment to a level above the ambient barometricpressure; decreasing the pressure of the reduced oxygen environment to alevel below the ambient barometric pressure; and repeating the cycle ofincreasing and decreasing environmental pressure above and below theambient pressure multiple times.
 132. A method as recited in claim 128,wherein said radio frequency field is an oscillating radio frequencyfield with a field strength of greater than approximately 3 kV/cm. 133.A method as recited in claim 128, wherein said ballast gas comprisesnitrogen gas, carbon dioxide gas or a combination of nitrogen gas andcarbon dioxide gases.
 134. A method as recited in claim 128, whereinsaid chemicals comprise a chemical from the group of chemicalsconsisting essentially of an alcohol, an insecticide, an oxygen radical,ozone, hydrogen peroxide, the nitrogen oxides group of compounds; andthe sulfur oxides group of compounds.
 135. A method for insect and mitecontrol, comprising: depriving an insect or mite of oxygen; andintroducing metabolic stress in said insect or mite while in an oxygendeprived state by subjecting said insects or mites to an oscillatingradiofrequency field.
 136. A method as recited in claim 135, furthercomprising: exposing said insect or mite to a chemical selected from thegroup of chemicals consisting essentially of an alcohol, an insecticide,an oxygen radical, ozone, hydrogen peroxide, the nitrogen oxides groupof compounds; and the sulfur oxides group of compounds.